In studying the electrostatic interaction of hemoglobin and 2,3- diphosphoglycerate (DPG), DPG itself cannot be used very effectively because of the high pK of some of its ionizable groups. The use of DPG analogs is proposed, represented by aliphatic or aromatic polyacids where carboxyl or sulfonic or other strong acidic groups are present. A preliminary investigation of the capability of polycarboxylic acids of decreasing the oxygen affinity of human hemoglobin gave very encouraging results. Their ionization is complete at pH 7, so they can be used to study the additional Bohr effect produced in the near alkaline region by their interaction with hemoglobin. This will be carried out essentially on the basis of acid-base titrations, which will make possible to measure the number and pK shifts of the hemoglobin side chains involved in the interaction, and the overall affinity constant with its pH dependency of hemoglobin for the effector. The use of different hemoglobins will help in the identification of the side chains involved in the interaction, and the use of different DPG analogs will help in deciding if there is or not a common mechanism for the interaction. The additional Bohr effect produced by the interaction of hemoglobin and its effectors can also be used to measure the correlation between conformational changes and ligand binding in the hemoglobin system. In fact the fraction of additional Bohr protons released when hemoglobin is partially oxygenated in presence of an effector can be taken as the measure of the amount of oxy conformation produced in the system by the presence of ligand. Extreme care will be exerted in measuring the fractional saturation with ligands correspondent to the fraction of additional Bohr protons liberated.